Project Summary: Gram-negative bacteria harboring carbapenemases are rapidly spreading worldwide and are a significant public health concern, resulting in mortality upwards of 60%. Carbapenemase-producing organisms (CPO) are considered a triple threat due to their increasing prevalence, their multidrug-resistant (MDR) profile, and their ease of transmission from species to species and even among different genera of Gram-negative bacteria through transmissible genetic elements (i.e. transposons, insertion sequences, and plasmids). Knowledge gaps: Studies of CPO transmission in the US have generally been limited to outbreak settings; data regarding transmission in endemic settings are largely unknown. Additionally, studies evaluating transmission of carbapenemases have generally been restricted to Enterobacteriaceae; The role of glucose non-fermenters (e.g., Pseudomonas and Acinetobacter spp.) has mostly been ignored. We propose to fill these gaps in knowledge by understanding the molecular mechanisms of antibiotic resistance and spread amongst all CPO (Enterobacteriaceae and glucose non-fermenters) in a region of the US endemic for CPO. These data will be critical in determining how best to limit CPO transmission between patients and to guide therapeutic decisions. To accomplish these goals, we have assembled a team of clinician-scientists, biomedical engineers, and bioinformaticians with expertise in clinical microbiology, epidemiology, infectious diseases, antibiotic stewardship, and in applications of the novel MinION technology. Aim 1: Determine the molecular mechanisms of spread involved in the dissemination of CPO in a non-outbreak setting using whole genome sequencing (WGS). Aim 2: Identify all antibiotic-resistance genes harbored by the CPO isolates to link the resistance genes to the circulating clones or plasmids harboring carbapenemases. Aim 3: Determine if the association of resistance genes with particular plasmids or clones can be used to accurately predict antibiotic susceptibility profiles of CPO clinical isolates. Significance and Impact: By gaining a better understanding of the complexity of the molecular mechanisms of resistance and spread among CPO, we can help guide therapy and appropriate prevention strategies to halt the spread of these important pathogens within the clinical setting.